Cells of the immune system arise from pluripotent stem cells through two main lines of differentiation: the lymphoid lineage that produces lymphocytes (T cells and B cells) and the myeloid lineage that produces phagocytes (monocytes, macrophages and neutrophils) and other cells. T cells and B cells are produced at a high rate (approximately 109 per day) in the primary or central lymphoid organs, i.e., the thymus and bone marrow, respectively. These lymphocytes can migrate via the blood circulation into the secondary lymphoid organs (spleen, lymph nodes, tonsils, and mucosa-associated lymphoid tissue). B cells, or B lymphocytes, represent about 5 to 15% of the circulating lymphoid pool, and are classically defined by the presence of immunoglobulin molecules on their surface membrane. These immunoglobulin molecules are produced by the B cells themselves and are inserted into the surface membrane where they act as specific antigen receptors.[1, 2] Upon activation, B cells combat extracellular pathogens and their products by releasing immunoglobulins, which act as antibodies that specifically recognize and bind to a particular target molecule, called the antigen.
B-cell development, also known as B lymphopoiesis, in mouse and in man can be divided into two main phases, an antigen-independent phase of fresh production of precursor B cells in the bone marrow that mature into functional B lymphocytes and an antigen-dependent phase, in which the mature B lymphocyte compartment is maintained by regeneration, turnover and selection processes.[3] Once the immune system has been built, it contains around 5×108 and 1012 cells of the B lymphocyte lineage in the mouse and in man, respectively, of which 5 to 10% are precursor B cells that are active in continuous production of fresh B cells, whereas over 90% are mature B cells.
The many different B cells of the immune system each produce different immunoglobulin (Ig) molecules, which can specifically bind to a foreign antigen. These Ig molecules consist of two identical Ig heavy chains and two identical Ig light chains, Igκ or Igλ. The antigen-binding variable domains of the Ig chains differ per B cell and are encoded by different combinations of variable (V), diversity (D), and joining (J) gene segments in the case of Ig heavy chains and different combinations of V and J gene segments in the case of Igκ and Igλ chains.[4, 5] The many different V, D and J gene segments in the Ig heavy chains (IGH) gene and the many different V and J gene segments of the Igκ (IGK) and Igλ (IGL) genes determine the potential V(D)J combinatorial repertoire, which is estimated to consist of >2×106 different Ig molecules in man (FIG. 1). During B-cell development in the bone marrow, precursor B cells form specific exons for the variable domains of antibody molecules by recombining individual V, D, and J gene segments via so-called gene rearrangement processes.[4] For example, in the IGH gene D to J rearrangement generally occur before V to D-J rearrangement, resulting in a specific V-D-J exon that can be transcribed into IGH mRNA and translated into IgH protein chains (FIG. 2). Comparable V-(D-)J rearrangement processes occur in IGK and IGL genes as well as in the T-cell receptor (TCR) genes, which encode the antigen-recognizing TCR molecules of T cells. [4, 6]
All V, D, and J gene segments are flanked by specific homologous recombination signal sequences (RSS).[7] These RSS consist of a conserved palindromic heptamer sequence (CACAGTG) adjacent to the coding sequence and a conserved nonamer sequence (ACAAAAACC) that are separated by a less conserved spacer region of either 12 or 23 base pairs (bp). In principle, only RSS of different spacer length join efficiently, known as the so-called 12/23 rule (FIG. 3). However, sometimes incomplete RSS, only consisting of a heptamer, are used. RSS are recognized by recombination activation gene 1 and 2 proteins (RAG1 and RAG2), which are able to cleave the DNA between the heptamer and the coding end of the involved gene segment.[8,9 ] The DNA cleavage results in a hairpinned coding end and a blunt 5-phosphorylated signal end. A so-called coding joint is obtained after cleavage and ligation of the hairpinned coding ends. During this ligation process, further (junctional) diversity of the coding joints is obtained by deletion and insertion of nucleotides, resulting in a highly diverse junctional region.[10] The V-(D-)J exon with the junctional region together with the constant exons are transcribed into mRNA and translated into protein (FIG. 2). The signal ends are also ligated and thereby form an extrachromosomal circular excision product containing the two coupled RSS, which is referred to as the signal joint (FIG. 2).
The extrachromosomal (episomal) excision product of the Ig gene rearrangement is also called “B-cell receptor excision circle” (BREC). These episomal products cannot replicate in the cell and appear to be highly stable structures, which can persist for a significant length of time. Consequently, BRECs can be found not only in precursor B cells but also in mature B lymphocytes. The role of the excision products in mature B cells is not fully clear.
During B-cell differentiation in bone marrow, the IGH genes and one of the Ig light chain genes (IGK or IGL) have to rearrange functionally in order to produce a complete Ig molecule. IGH gene rearrangements (D to J, followed by V to D-J) precede the Ig light chain gene rearrangements with IGK gene rearrangements occurring prior to IGL gene rearrangements (FIG. 4).[11, 12] Functional IGK gene rearrangements result in Igκ producing B cells that usually retain their IGL genes in germline configuration.[13, 14] If the IGK gene rearrangements are not functional, the IGL genes rearrange in an attempt to produce Igλ+ B cells. Interestingly, most Igλ-producing B cells have deleted their IGK genes on at least one allele, generally on both alleles (FIG. 4).[14, 15]
IGK gene deletions are mediated via rearrangement of the so-called kappa-deleting element (Kde), which is located approximately 24 kb downstream of the constant (C) kappa gene segment (Cκ).[16-19] Kde can either rearrange to a heptamer RSS in the intron between the Jκ and Cκ gene segments (intronRSS) or to one of the available Vκ gene segments (FIG. 5). The intronRSS-Kde rearrangement deletes the Cκ gene segment, whereas Vκ-Kde rearrangements delete the complete Jκ-Cκ region. Both types of Kde rearrangements also delete the two enhancers of the IGK locus (iEκ and 3′Eκ) implying that Kde rearrangements are “end-stage” rearrangements, precluding any further rearrangement in the IGK locus (FIG. 5).[20] The Kde rearrangements are found in precursor B cells and in mature B cells, particularly in (virtually) all Igλ+ mature B cells.[21-23] Also, a part of the Igκ+ B cells contain Kde rearrangements, but they are absent in the majority of Igκ+ B cells.[14, 22]
It will be understood that B-cell development is important during health and disease. Dysfunction of the precursor B-cell compartment or the mature B-cell compartment is observed in various types of immune diseases, during immunosuppressive treatments (e.g., with cyclosporin), during cancer treatment,[24, 25] and following bone marrow transplantation (BMT).[26] Furthermore, B-cell development is typically reduced during aging.[27]
BMT and peripheral blood stem cell transplantation (PBSCT) are procedures that aim at restoration of the stem cell compartment, when it is affected by specific diseases (e.g., primary immunodeficiencies, cancer, etc.) and/or when it has been destroyed by high doses of chemotherapy and/or radiation therapy. Generally speaking, the goal is to replace the diseased marrow with healthy bone marrow. Bone marrow is mainly concentrated in the skull, ribs, sternum, vertebrae and pelvic bone, and less so in other bones. It contains immature hematopoietic cells called hematopoietic stem cells that produce blood cells. Most stem cells are found in the bone marrow, but some stem cells called peripheral blood stem cells (PBSCs) can be found in the bloodstream. Stem cells can divide to form more stem cells, or they can mature into white blood cells, red blood cells, or platelets. In cancer treatment, the main purpose of BMT and PBSCT is to make it possible for patients to receive very high doses of chemotherapy and/or radiation therapy. Chemotherapy and radiation therapy generally affect cells that divide rapidly, including bone marrow cells. BMT and PBSCT replace stem cells that were destroyed by treatment. The healthy, transplanted stem cells can restore the bone marrow's ability to produce the blood cells the patient needs. After entering the bloodstream, the transplanted cells travel to the bone marrow, where they begin to produce new white blood cells, red blood cells, and platelets in a process known as “engraftment.”
A major problem in the field of transplantation, be it BMT or PBSCT, is the difficulty to monitor the efficacy of transplantation and, herewith, to determine the optimal treatment protocol. Evaluation of B-cell-dependent antibody production is one way to determine how well the new bone marrow is working. However, it is very difficult to obtain direct insight into the origin of B cells in the transplanted recipient. This is because after BMT or PBSCT, B cells can, in theory, regenerate from several sources: (1) mature B cells of the transplant recipient which survived the pre-transplantation chemotherapeutic intensification treatment; such cells may be seeded in the bone marrow, lymph nodes, or spleen; (2) mature B cells present in the graft; (3) hematopoietic stem cell progenitors in the transplant that differentiate after grafting in the recipient; and (4) residual recipient stem cells. Thus, given the various possible B cell sources, serotyping does not allow discrimination between antibodies produced by newly developed B cells and antibodies produced by old mature B cells that have expanded in the periphery of the recipient. If the antibody production is solely based on expanding mature B cells (not on newly produced B cells as well), antibody production will end . as the old B cells die off. Rather, the efficacy of regeneration of the precursor B-cell compartment should ideally be monitored by determining the “age” of the B cells present in a subject, allowing distinguishing between newly produced B cells and “old” B cells.